Gene knockout technology in mice has been instrumental in investigating gene functions in mammals. It has been used to reveal in vivo functions of genes in normal as well as pathogenic pathways. However, technical complexities, length of time and high cost have limited its use in a wider variety of scientific inquiries. RNA interference (RNAi) can mediate sequence-selective suppression of gene expression in a wide variety of eukaryotes by introducing short RNA duplexes (called small interfering RNAs or siRNAs) with sequence homologies to the target gene. Recent experiments indicate that small hairpin RNAs (shRNAs) transcribed in vivo can trigger degradation of corresponding mRNAs similar to siRNA and achieve effective knockdowns in cultured cells, and in some instances, in vivo. Despite these advances, the current RNAi technology can only achieve gene knockdown but not gene "knockout". Therefore, whether RNAi can serve as an alternative to gene knockout technology to generate animal disease models and to investigate molecular pathways in vivo is unclear. I propose to develop the RNAi technology that can mimic knockout conditions in vivo and may serve as an alternative to the conventional gene knockout. The central idea is to increase the potency of RNAi so that it can achieve virtual knockout. The R21 phase has three aims: (1) develop strategies that will increase the RNAi potency so that it mimics the condition of a knockout using cell cultures, (2) apply the most effective strategy that are defined in aim 1 to "knockout" genes that are associated with aging diseases in transgenic mice and to determine whether animal models for these diseases can be generated, and (3) develop inducible promoters for shRNA synthesis so that inhibition of specific gene expression can be controlled spatially and temporally. If successful, this technology can be widely applied as an alternative to gene knockout technology for investigation of gene functions in vivo and generation of animal disease models.